Background

Oxidative stress is one of the important mediators of vascular complications in diabetes including nephropathy. High glucose (HG) generates reactive oxygen species (ROS) as a result of glucose auto-oxidation, metabolism, and formation of advanced glycosylation end products. The concept of ROS-induced tissue injury has recently been revised with the appreciation of new roles for ROS in signaling pathways and gene expression.

Methods and Results. High glucose rapidly generated dichlorofluorescein-sensitive cytosolic ROS in rat and mouse mesangial cells. Neither L-glucose nor 3-O-methyl-D-glucose increased cytosolic ROS and cytochalasin B, an inhibitor of glucose transporter, effectively inhibited HG-induced ROS generation, suggesting that glucose uptake and subsequent metabolism are required in HG-induced cytosolic ROS generation. H₂O₂ up-regulated fibronectin mRNA expression and protein synthesis; this up-regulation was effectively inhibited by protein kinase C (PKC) inhibitor or by depletion of PKC. The HG-induced generation of ROS was, in turn, related to activation of PKC and transcription factors nuclear factor-B (NF-B) and activator protein-1 (AP-1) as well as to the up-regulation of transforming growth factor-1 (TGF-1), fibronectin mRNA expression and protein synthesis, because antioxidants effectively inhibited HG-induced PKC, NF-B, AP-1 activation, and TGF-1 and fibronectin expression in mesangial cells cultured under HG.

Conclusions

Although signal transduction pathways linking HG, ROS, PKC, transcription factors, and extracellular matrix (ECM) protein synthesis in mesangial cells have not been fully elucidated, the current data provide evidence that ROS generated by glucose metabolism may act as integral signaling molecules under HG as in other membrane receptor signaling.

GENERATION OF REACTIVE OXYGEN SPECIES IN MESANGIAL CELLS CULTURED UNDER HIGH GLUCOSE

We recently visualized dichlorofluorescein (DCF)-sensitive ROS generation in rat and mouse mesangial cells cultured under HG by a confocal microscopy as shown in Figure 1 (abstract; Ha et al, J Am Soc Nephrol 10:A3452, 1999). High glucose induced DCF-sensitive cytosolic ROS in as early as 15 minutes and gradually increased up to 4 hours, in contrast to control glucose. Unlike D-glucose, neither 25 mmol/L L-glucose nor 3-O-methyl-D-glucose induced cytosolic ROS generation. In addition, a glucose transporter inhibitor cytochalasin B effectively inhibited HG-induced cytosolic ROS. These results suggest that glucose uptake and subsequent metabolism, but not glucose auto-oxidation, are required for HG-induced ROS generation in mesangial cells. 3-O-methyl-D-glucose is a glucose analog that enters into the cell but is not metabolized. Lack of effect of 3-O-methyl-D-glucose on cytosolic ROS generation in rat and mouse mesangial cells is different from the observation in human endothelial cells³⁰, suggesting that different regulatory mechanisms may operate in HG-induced ROS generation in different cell types or species. Inasmuch as dichlorofluorescin diacetate enters into cells and produces DCF, which fluoresces when it reacts with hydroperoxides or H₂O₂^31,32 and given that catalase effectively inhibits HG-induced ROS generation, H₂O₂ appears to be the main ROS generated by HG in mesangial cells. Catalase is a 60 kD protein that forms a homodimer in solution and thus does not simply diffuse into cells. Energy-dependent and perhaps receptor-mediated processes have been suggested in cellular uptake of catalase in vascular smooth muscle cells²¹. HG-induced H₂O₂ generation agrees with the conclusions of a previous study that demonstrated increased H₂O₂ in mesangial cells cultured under HG³³. At present, little is known about the mechanisms of HG-induced H₂O₂ generation or target molecules of H₂O₂. These areas require future studies.

Figure 1.

HG generates dichlorofluorescein-sensitive reactive oxygen species in mesangial cells. Synchronized quiescent mesangial cells grown on coverglass were incubated with 5.6 or 30 mmol/L glucose for 1 hour or with 100 mol/L H₂O₂ for 15 min, washed with Dulbecco's phosphate-buffered saline and incubated in the dark for 5 minutes in Krebs-Ringer solution containing 5 mmol/L 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Culture dishes were transferred to a Leica DM IRB/E inverted microscope, equipped with 20 Fluotar objective and Leica TCS NT confocal attachment and the ROS generation was visualized.

Full figure and legend (66K)

Advanced glycation end products (AGE) are produced by a process involving nonenzymatic modification of proteins by physiologic sugars and their reactive dicarbonyls such as glyoxal, 3-deoxyglucosone, and methylglyoxal and are independent risk factors for diabetic nephropathy^34,35,36. In addition to altered protein structure and function induced by AGE, AGE bind to cell surface receptors and initiate ROS production, which activates expression of gene products including cytokines and hormones^37,38. Although the production of ROS in mesangial cells in response to AGE has not been demonstrated, HG may generate cytosolic ROS directly through glucose metabolism and indirectly through AGE.

H₂O₂-INDUCED FIBRONECTIN UP-REGULATION IN MESANGIAL CELLS

HG-induced ROS generation appears to play a major role in mesangial ECM expansion, which is the major pathologic feature in diabetic nephropathy³⁹, because HG-induced collagen production in cultured rat mesangial cells was effectively prevented by two antioxidants, taurine⁶ and vitamin E⁷. We also demonstrated that rebamipide and dimethylthiourea at concentrations inhibiting HG-induced lipid peroxidation blocked fibronectin mRNA expression and protein synthesis by mouse mesangial cells cultured under HG⁸. In addition, we earlier reported that H₂O₂ at 100 mol/L significantly increased fibronectin mRNA expression and protein synthesis by murine mesangial cells as shown in Figure 2 (abstract; Ha and Kim, Nephrology 3(Suppl 1):A736, 1997).

Figure 2.

H₂O₂ up-regulates fibronectin mRNA expression and protein synthesis in mesangial cells through PKC activation. Synchronized quiescent mesangial cells were stimulated with 30 mmol/L glucose, 100 mol/L H₂O₂, or 100 nmol/L phorbol ester (PMA) in the presence or absence of PKC inhibition. Calphostin C (100 nmol/L) was used to inhibit PKC. Endogenous PKC was depleted by preincubating cells with 100 nmol/L PMA for 24 hours. (A) After 24 hours' incubation for a given experimental condition, total RNA was isolated, electrophoresed, and transferred onto nylon membranes. Northern blots were hybridized with ³²P-labeled cDNA probes for fibronectin (FN) and glyceraldehyde-3-phosphate dehydrogenase. Symbols are: , no calphostin C; , 100 nmol/L calphostin C. (B) Aliquots of mesangial cell–conditioned media were electrophoresed under reducing conditions and transferred onto nitrocellulose membranes and Western blots were performed using the ECL detection system. Autoradiographs were developed by exposing blots to radiographic films. Values were expressed as mean standard error of four experiments. Symbols are: , no PKC depletion; , PKC depletion. *P < 0.05 compared with control without PKC inhibition. †P < 0.05 compared with the corresponding values without PKC inhibition.

Full figure and legend (58K)

TARGET MOLECULES ON WHICH HIGH GLUCOSE-INDUCED REACTIVE OXYGEN SPECIES ACT TO PROPAGATE THE SIGNAL IN MESANGIAL CELLS

As has been reviewed extensively^14,40,41, the growing medical literature from disciplines outside of diabetes research show that ROS can activate most known signal transduction pathways. In diabetes, protein kinase C (PKC)⁴² and, more recently, mitogen-activated protein kinases (MAPK)^43,44 have been shown to be activated and mediate HG-induced tissue injury. In addition, HG may transduce messages to the nucleus leading to gene transcription by a class of proteins called transcription factors that bind to specific DNA sequences to regulate RNA polymerase II activity. NF-B and activator protein (AP)-1 are well known redox sensitive transcription factors that activate the genes coding for cytokines, growth factors, and ECM proteins^40,41. Target molecules on which ROS act to propagate the signal determine the cell type–specific and diverse events and outcomes.

Activation of PKC is one of the major mechanisms involved in HG-induced glomerular injury⁴² and produces ROS and subsequent lipid peroxidation^45,46,47. Conversely, ROS also activate PKC. H₂O₂ activates PKC through direct or indirect activation of phospholipase D, which hydrolyzes phosphatidylcholine to produce diacylglycerol (DAG)⁴⁸, and through de novo synthesis of DAG resulting from inhibition of glyceraldehyde phosphate dehydrogenase⁴⁹. In addition, activation of PKC is sensitively regulated through redox changes in sulfhydryl groups of cysteine-rich regions of PKC^50,51. All these observations suggest facilitative interaction between ROS and PKC under hyperglycemia. In fact, structurally different antioxidants suppressed an increase in PKC in rat mesangial cells⁵². We have reported that inhibition of PKC effectively blocked not only phorbol ester-induced but also HG-and H₂O₂-induced fibronectin mRNA expression and protein synthesis by mesangial cells as shown in Figure 2.

MAPK consist of groups of serine–specific kinases that are activated by extracellular stimuli through dual phosphorylation at conserved threonine and tyrosine residues. Three major groups of MAPK exist; the extracellular signal-regulated kinases (ERK), the c-Jun N-terminal kinases (JNK), and the p38 kinases. Understanding of their roles in cell physiology continues to evolve and all three MAPK play essential roles in the signal transduction of many biologic events such as cell growth, differentiation, and apoptosis. Tomlinson et al⁴³ recently proposed MAPK as glucose transducers for diabetic complications. Activation of an MAPK cascade in diabetic glomeruli and mesangial cells cultured under HG has been reported⁴⁴. Given that MAPK activation was observed 5 days after stimulation with HG in the study cited, other HG-induced cytokines and growth factors in addition to HG itself may have augmented MAPK activation. For example, TGF-1, the final mediator of HG-induced ECM accumulation⁵³, also uses MAPK as its signaling pathway as reviewed by Choi et al and Kikkawa elsewhere in this Supplement. Because ROS are the major stimuli activating MAPK in various cells^40,41,54, it would be interesting to know whether antioxidants can prevent hyperglycemia-induced MAPK activation.

Because it remains unclear which transcription factors are ultimately activated by HG-induced ROS generation and thereby lead to diabetic glomerular injury, we examined whether HG-induced ROS activate NF-B and AP-1 by electrophoretic mobility shift assay (EMSA). NF-B is the first eukaryotic transcription factor shown to respond directly to ROS^55,56. This transcription factor plays an important role in the regulation of genes involved in immune and inflammatory process. AP-1, a heterodimer of Fos and Jun proteins, controls genes involved in cell growth. Previous studies demonstrated that AP-1 is activated by ROS^40,41. It is possible that the essential Cys residues of NF-B, c-Fos, and c-Jun are targets of ROS modification, so that this modification, together with phosphorylation events, may be responsible for their signaling mechanisms¹⁴.

As has been reported in endothelial and vascular smooth muscle cells^30,57,58,59, D-glucose induced NF-B activation in mesangial cells in a dose- and time-dependent manner and antioxidants effectively inhibited HG-induced NF-B activation, which suggests ROS as mediators of HG-induced activation of transcription factors (abstract; Ha et al, J Am Soc Nephrol 9:A3226, 1998). 30 mmol/L D-glucose but not L-glucose rapidly (i.e., less than 15 minutes) and continuously (up to 48 hours) activated AP-1 compared with 5.6 mmol/L (control) glucose (Figure 3a,B). In addition, H₂O₂ activated both NF-B (data not shown) and AP-1 Figure 3c in a dose-dependent manner.

Figure 3.

Reactive oxygen species activate activator protein-1 in mesangial cells cultured under high glucose. Synchronized quiescent mesangial cells were stimulated under given experimental conditions. Nuclear proteins were prepared and subjected to EMSA. (A) Representative EMSA showing mesangial AP-1 activation by D-glucose but not by L-glucose. Mesangial cells were stimulated for 1 hour. (B) Time response of mesangial AP-1 activation. Mesangial cells were incubated with 30 mmol/L (high) glucose or 5.6 mmol/L (control) glucose for indicated periods. (C) Effects of H₂O₂ on AP-1 activation in mesangial cells.

Full figure and legend (96K)

The biological significance of NF-B activation in MC under HG, however, is still not known, and further studies are required. HG-induced NF-B activation may be involved in HG-induced monocyte chemotactic peptide (MCP)-1 mRNA expression observed in human mesangial cells⁶⁰. The promotor region of MCP-1 contains an NF-B binding site⁶¹ and MCP-1 mRNA expression is regulated by NF-B activation in mesangial cells⁶². In addition, Smad 7, an inhibitor of autoinduction of TGF-, is transcriptionally activated by p65 subunit of NF-B as reviewed by Böttinger in this Supplement.

HG-induced AP-1 activation demonstrated by EMSA agrees with previous studies that reported increased expression of growth-related proto-oncogene c-fos and c-jun in mesangial cells cultured under HG⁶³ and in the glomeruli from streptozotocin-induced diabetic rats⁶⁴. The c-Fos and c-Jun proteins, along with other Jun proteins, such as JunB and JunD, are members of the AP-1 transcription factor family⁶⁵ and a heterodimeric interaction between the two allows for binding to consensus DNA sequences in the regulatory elements of certain genes, called AP-1 and TPA responsive sites⁶⁶. Because AP-1 consensus sites exist in the promotor regions of TGF-1⁶⁷, fibronectin⁶³, and laminin B genes⁶³, it is possible that cytosolic ROS may up-regulate TGF-1, fibronectin, and laminin B expression through AP-1 activation in mesangial cells cultured under HG. However, the relationships among ROS, AP-1 activity, and ECM production under HG have not been fully defined and require future studies.

AMELIORATION OF HIGH GLUCOSE-INDUCED ALTERATIONS IN MESANGIAL CELL BIOLOGY BY ANTIOXIDANTS

As summarized earlier in this paper, antioxidants effectively prevented altered biology of mesangial cells cultured under HG. For example, antioxidants abolished HG-induced cytosolic ROS generation (abstract; Ha et al, J Am Soc Nephrol 10:A3452, 1999), PKC activation⁵², up-regulations of TGF-1^8,52 and fibronectin⁸, and activation of transcription factors NF-B (abstract; Ha et al, J Am Soc Nephrol 9:A3226, 1998). These observations provide the final evidence that ROS may act as signaling molecules.

CONCLUSION

In the past, large doses of ROS were regarded as toxic to cells. This concept has recently been revised in light of new roles for ROS in signaling and gene expression. New data support a role for ROS as second messenger molecules in nonphagocyte cells. As discussed in this paper, ROS generated by accelerated glucose metabolism may also act as integral signaling molecules under HG as in other membrane receptor signaling Figure 4. However, cell-type specificity and diversity must exist in signaling events and outcomes by which cells respond to HG-induced ROS. This may explain the different cell responses such as proliferation, differentiation, apoptosis, and ECM accumulation in different cell types. Thus, further studies to identify target molecules on which ROS act to propagate the signal in mesangial cells under HG will help us understand the mechanisms involved in HG-induced mesangial cell activation and develop strategies for rational treatment of diabetic nephropathy.

Figure 4.

Proposed role of reactive oxygen species as glucose signaling molecules in mesangial cells. HG may increase cytosolic ROS directly through its metabolism and indirectly through AGE formation. Facilitative interaction occurs between ROS and PKC in hyperglycemia. ROS and PKC may activate MAPK cascade and several transcription factors including NF-B and AP-1. It is also speculated that the essential Cys residues of NF-B, c-Fos, and c-Jun can be modified by ROS and this modification together with phosphorylation events may be responsible for their signaling mechanisms.

Full figure and legend (22K)

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Acknowledgments

Original works from the authors are supported by grants from Korea Science and Engineering Foundation (KOSEF 981–0714–106–2 and 986–0700–003–2).